Project Summary Tissue stem cells provide for the maintenance and regeneration of organs and tissues throughout life. The ability of stem cells to contribute to tissue homeostasis depends on their unique ability to generate new stem cells (self-renewal), as well as specialized cell types (differentiation). Stem cell behavior is influenced by the integration of intrinsic factors with extrinsic cues provided by the local microenvironment (or ?niche?) and circulating, systemic factors. Thus, identification and characterization of the mechanisms that are involved in regulating stem cell behavior holds significant promise for the maintenance, manipulation and expansion of stem cells for use in regenerative medicine. The capacity of stem cells to self-renew or differentiate has been attributed to distinct metabolic states. Our lab recently found that an increase in triacylglycerides (TAG) in the form of lipid droplets (LDs), either through disruption of fatty acid oxidation (FAO) or stimulation of lipid anabolism, correlates with a loss of germline stem cell (GSC) fate in the Drosophila testis. Genetic or pharmacologic enhancement of lipid catabolism rescued the loss of GSCs, indicating that GSCs are sensitive to the levels of intracellular lipids. Here, we propose to identify the mechanism(s) by which ectopic lipid accumulation leads to loss of GSC identity and to use the well- characterized Drosophila male germline as a model to uncover mechanisms used to tightly regulate lipid metabolism in GSCs to maintain tissue homeostasis through the following Specific Aims: Aim 1: To characterize mechanisms by which increased intracellular lipids contribute to loss of GSCs Aim 2: To investigate the role of phosphatidic acid (PA) metabolism in the Drosophila testis Aim 3: To characterize the role of the phosphatidic acid phosphatase (PAP) dLipin in the Drosophila testis Taken together, these Aims will contribute to a better understanding of how lipid metabolism can influence tissue homeostasis by influencing stem cell behavior. Furthermore, our work will provide a genetic model to characterize mechanisms that cells in non-adipose tissues use to manage ectopic lipids to avoid lipotoxicity. Finally, our findings will provide important insights into how stem cell and/or germ cell function may be altered in individuals suffering from metabolic disorders, such as obesity and/or type 2 diabetes, leading to strategies to regulate stem cell function, in vivo, in patients by modulating metabolic pathways pharmacologically or through diet.